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Moreno, Marta; Saavedra, Marlon P; Bickersmith, Sara A; Prussing, Catharine; Michalski,Adrian; Tong Rios, Carlos; Vinetz, Joseph M; Conn, Jan E; (2017) Intensive trapping ofblood-fed Anopheles darlingi in Amazonian Peru reveals unexpectedly high proportions of avianblood-meals. PLoS neglected tropical diseases, 11 (2). e0005337. ISSN 1935-2727 DOI:https://doi.org/10.1371/journal.pntd.0005337

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RESEARCH ARTICLE

Intensive trapping of blood-fed Anopheles

darlingi in Amazonian Peru reveals

unexpectedly high proportions of avian

blood-meals

Marta Moreno1*, Marlon P. Saavedra2, Sara A. Bickersmith3, Catharine Prussing4,

Adrian Michalski3, Carlos Tong Rios2, Joseph M. Vinetz1, Jan E. Conn3,4

1 Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla,

California, United States of America, 2 Laboratorio ICEMR-Amazonia, Laboratorios de Investigacion y

Desarrollo, Facultad de Ciencias y Filosofia, Universidad Peruana Cayetano Heredia, Lima, Peru,

3 Wadsworth Center, New York State Department of Health, Albany, NY, United States of America,

4 Department of Biomedical Sciences, School of Public Health, State University of New York-Albany, NY,

United States of America

* [email protected]

Abstract

Anopheles darlingi, the main malaria vector in the Neotropics, has been considered to be

highly anthropophilic. However, many behavioral aspects of this species remain unknown,

such as the range of blood-meal sources. Barrier screens were used to collect resting

Anopheles darlingi mosquitoes from 2013 to 2015 in three riverine localities (Lupuna,

Cahuide and Santa Emilia) in Amazonian Peru. Overall, the Human Blood Index (HBI) ran-

ged from 0.58–0.87, with no significant variation among years or sites. Blood-meal analysis

revealed that humans are the most common blood source, followed by avian hosts (Galli-

formes-chickens and turkeys), and human/Galliforme mixed-meals. The Forage Ratio and

Selection Index both show a strong preference for Galliformes over humans in blood-fed

mosquitoes. Our data show that 30% of An. darlingi fed on more than one host, including

combinations of dogs, pigs, goats and rats. There appears to be a pattern of host choice in

An. darlingi, with varying proportions of mosquitoes feeding only on humans, only on Galli-

formes and some taking mixed-meals of blood (human plus Galliforme), which was detected

in the three sites in different years, indicating that there could be a structure to these popula-

tions based on blood-feeding preferences. Mosquito age, estimated in two localities, Lupuna

and Cahuide, ranged widely between sites and years. This variation may reflect the range of

local environmental factors that influence longevity or possibly potential changes in the abil-

ity of the mosquito to transmit the parasite. Of 6,204 resting An. darlingi tested for Plasmo-

dium infection, 0.42% were infected with P. vivax. This study provides evidence for the first

time of the usefulness of barrier screens for the collection of blood-fed resting mosquitoes to

calculate the Human Blood Index (HBI) and other blood-meal sources in a neotropical

malaria endemic setting.

PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005337 February 23, 2017 1 / 19

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OPENACCESS

Citation: Moreno M, Saavedra MP, Bickersmith

SA, Prussing C, Michalski A, Tong Rios C, et al.

(2017) Intensive trapping of blood-fed Anopheles

darlingi in Amazonian Peru reveals unexpectedly

high proportions of avian blood-meals. PLoS Negl

Trop Dis 11(2): e0005337. doi:10.1371/journal.

pntd.0005337

Editor: Paulo Filemon Pimenta, Fundacao Oswaldo

Cruz, BRAZIL

Received: October 14, 2016

Accepted: January 18, 2017

Published: February 23, 2017

Copyright: © 2017 Moreno et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

files.

Funding: This research was funded by grant

U19AI089681 to JMV and grant AI110112 to JEC

from National Institutes of Health/National Institute

of Allergy and Infectious Diseases (www.niaid.nih.

gov). The Biodefense and Emerging Infectious

Disease training fellowship grant T32AI05532901

provided partial support for CP. The funders had no

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http://creativecommons.org/licenses/by/4.0/

http://www.niaid.nih.gov

http://www.niaid.nih.gov

Author summary

Anopheles darlingi is the major malaria vector in the Amazon. This species has been com-

monly described as highly anthropophilic throughout its geographic range, although little

is known about its feeding preferences. Scant information is available regarding the origin

of An. darlingi blood-meals. In the context of malaria elimination programs, the Human

Blood Index (HBI) may provide crucial information regarding mosquito-human contact

related to transmission dynamics. Additionally, collection of resting An. darlingi is chal-

lenging, mainly because the resting behavior of this species has not been well character-

ized. Our study, conducted from 2013–2015 in three localities in Loreto Department in

the Peruvian Amazon, showed for the first time the efficacy of the barrier screen method-

ology for collecting recently blood-fed An. darlingi in a neotropical setting for the purpose

of identifying the source of their blood-meals. Our data show that An. darlingi feeds on

humans, Galliformes, dogs, pigs and goats, and that 30% of the mosquitoes fed on more

than one type of host. Despite this opportunistic feeding behavior, however, An. darlingiis primarily anthropophilic. We hypothesize that mosquito population structure is associ-

ated with feeding preferences, which may affect the pattern of malaria transmission in the

area.

Introduction

The Human Blood Index (HBI), formerly known as the anthropophilic index or human blood

ratio, is the proportion of recently-fed mosquitoes, usually vector species that have taken a

human blood-meal [1]. This index is a very important component of the formulae used to

determine vectorial capacity and varies depending on mosquito species, collection area and

season or time of collection [2]. From an epidemiological standpoint, it is crucial to be able to

accurately identify mosquito blood-meals for studies of transmission dynamics of viral and

parasitic pathogens [3]. For example, in Equatorial Guinea, the calculation of this index before

and after indoor interventions to reduce malaria did not detect any mosquito behavioral dif-

ferences, and researchers concluded that control strategies in this region were ineffective [4].

In Central Kenya, anthropophily decreased in An. gambiae after the introduction of long last-

ing insecticide nets (LLINs) and zooprophylaxis [5]. However, in southern Zambia, after two

years of LLIN intervention, the main vector, Anopheles arabiensis, remained highly anthropo-

philic [6]. In Tanzania the HBI showed a change in the main blood-source in An. arabiensisbut not in An. funestus after the use of spatial repellent coils [7].

Another index to quantify host selection patterns is the incidence of multiple blood-meals

from the same host species (cryptic) or from two or more different host species (patent) [8].

Evidence that malarial mosquitoes take partial blood-meals from multiple hosts may be inter-

preted as interrupted blood-feedings that could increase the probability of both acquiring and

transmitting Plasmodium [9]. On the other hand, Burkot and colleagues [10] contend that

fewer gametocytes would be ingested per meal, resulting in lower mosquito infection rates.

Anopheles darlingi, the primary regional malaria vector in the Amazon Basin, is anthropo-

philic in the Iquitos region [11], although both human biting rate (HBR) and entomological

inoculation rate (EIR) vary widely [12] depending on the setting [13–15]. The An. darlingifeeding site in this region is exophagic and/or endophagic, depending on local circumstances

(e.g., vegetation cover, type of house) and host availability [11, 12, 14,15].

In 2015, Loreto Department reported 95% of the total malaria cases in Peru (59,349 of

62,220 total) with Plasmodium vivax as the most prevalent human parasite followed by P.

Anopheles darlingi host preference for avian blood in Amazonian Peru


role in study design, data collection and analysis,

decision to publish, or preparation of the

manuscript.

Competing Interests: The authors have declared

that no competing interests exist.

falciparum, with 46,924 and 12,425 cases, respectively [16]. Parker and collaborators [13] dem-

onstrated that high HBR, EIR, and infectivity of An. darlingi are a signature of remote riverine

malaria hot spots and hyperendemicity in certain areas of the Peruvian Amazon, upending

previous notions that transmission is hypoendemic throughout the peri-Iquitos region

[11,12]. Recent studies also detected very high seasonal HBR and moderate EIR in the peri-

Iquitos region [14, 15]. Most malaria cases occur during the rainy season, from December to

June [17] and a correlation was detected between An. darlingi abundance and peak river levels,

but there was no significant correlation between river level and malaria case numbers [12, 14,

15]. In this last study, mosquitoes positive for Plasmodium were collected in peridomestic

areas within approximately 10 m of the main house entrance, (a caveat being that very few An.

darlingi were found indoors despite extensive searching), suggesting that most malaria is trans-

mitted exophagically, where humans have little protection against mosquito bites.

Despite being the dominant malaria vector in Amazonia, few studies have documented the

blood-meal sources for An. darlingi. In Amapa state, Amazonian Brazil, an ELISA analysis

found that 13.1% of blood-meals were human; most resting An. darlingi had fed on cattle, pigs

and dogs [18]. Notwithstanding the relatively low level of HBI, these communities are endemic

for malaria, and An. darlingi is considered to be the most effective local vector [19]. In Peru,

no studies have been published on the identity of An. darlingi blood-meals, but potential non-

human hosts in rural residences near Iquitos include common peridomestic animals, dogs and

chickens, and several potential wild mammalian hosts [12].

Although resting mosquitoes are optimal for calculating HBI, adequate sample sizes can be

difficult to obtain in some habitats [18–20]. Little information exists on host preference and

resting behavior of An. darlingi. The location of resting sites of An. darlingi could be useful for

focal vector control if such mosquitoes are clustered non-randomly in the landscape. The

development of barrier screens as a method for collecting anophelines outdoors has been

tested successfully in the South East Pacific [20] and recently in southern Zambia [21].

This study was designed to address the following questions regarding An. darlingi feeding

behavior in the Peruvian Amazon: i) are barrier screens a useful tool to collect resting blood-

fed An. darlingi in the area; ii) what is the degree of anthropophily (HBI) in An. darlingi in con-

trast to more opportunistic behavior; iii) what is the influence of available host biomass and iv)

is there evidence of seasonal age-structure in An. darlingi.

Methods

Ethics statement

This study was approved by the Human Subjects Protection Program of the University of Cali-

fornia San Diego, La Jolla, California and by the Ethical Boards of Universidad Peruana Caye-

tano Heredia and Asociacion Benefica PRISMA, Lima, Peru.

Mosquito collections

The strategy of the barrier screen method of collecting mosquitoes outdoors is to intercept and

capture mosquitoes transiting between blood feeding and resting sites [20]. Two possible sce-

narios can be identified: 1) intercepting mosquitoes entering a village seeking a blood-meal

after emergence or oviposition; and 2) intercepting blood-fed mosquitoes leaving the village

and seeking resting sites for egg development (swamp, creek, stream, forest). In this Peruvian

study, barrier screens were placed to intercept mosquitoes flying between house-forest and

house-river depending on the specific characteristics of the locality. Mosquito collections were

performed in three villages in Loreto Department: Lupuna (LUP) and Cahuide (CAH) in the

peri-Iquitos area, and Santa Emilia (SEM), in a remote area ~150 km from Iquitos (Fig 1).



Detailed descriptions of these villages are in [15, 22]. In 2013, from March to May, a pilot

study was conducted using a single screen in LUP and CAH placed at different points within

each village (between the creek/river and village houses). Specimens were collected for 4 nights

(6PM- 6AM) each month.

Each barrier screen was constructed from a lightweight window screen mesh approximately

15 m long and 2 m high (S1 Fig). Screens were then attached to poles with thin wire. Permis-

sion from the inhabitants/owners was obtained prior to any activity, including setting up the

barrier screens and performing mosquito collections. Resting mosquitoes from the barrier

screens were sampled by manually searching the surface of the screens with a mouth aspirator

every hour for 15 minutes on each side, and the location (next to house, forest or river) and

height (> or < 1m above ground) of mosquitoes was recorded. Mosquitoes were captured and

stored by hour of collection and screen side separately. In 2014 (monthly) and 2015 (January-

June), the design was slightly modified to include four barrier screens in LUP and CAH to bet-

ter represent the An. darlingi population in each locality. When multiple screens were used per

village, data from each screen was maintained separately. In SEM, a remote village along the

Nahuapa River, collections were performed with two barrier screens for two nights in May-

June 2014 and May-September 2015. Additionally, in 2015, daytime mosquito collections

(6AM-6PM) with barrier screens were performed two days monthly from January-June in

LUP and CAH, and from May-July in SEM. Screen orientation, wind speed and direction

were recorded for every collection with a Windmate 300 Wind/Weather Meter. A census ques-

tionnaire of domestic hosts present in the study villages was performed in October 2014 in

LUP and CAH and May 2015 in SEM (S1 Table, Fig 2). Because the first study was performed

a year prior and the animal composition could have changed, the questionnaire included a ret-

rospective question to assess the presence of potential past hosts.

Fig 1. Map of the sites where the mosquito collections were performed in the Department of Loreto,

Amazonian Peru.

doi:10.1371/journal.pntd.0005337.g001



All specimens collected were morphologically identified using entomological keys [23–25]

and abdominal status recorded (unfed, blood-fed or gravid). Mosquitoes were stored and

labeled individually with silica gel and placed at 4˚C until subsequent analysis.

Estimation of parity and daily survival rate

To estimate the female age composition of the population, in March-April 2014 and February-

June 2015 in LUP and CAH a proportion of females were dissected to determine the parity

rates per hour, trap and side of trap [26]. Parity is also used as an indicator of mosquito sur-

vival under natural conditions. Mosquito longevity (life expectancy) was estimated using

Davidson’s methodology (1954) Age ¼ 1

log‘P, where ‘ is the natural logarithm of the constant P

(daily survival rate). (P) was calculated P =ffiffiffiffiffiffiPRgcp

, where PR is the ratio of parous mosquitoes

and the total number of females dissected, and gc is the duration of the gonotrophic cycle in

days [27]. A limitation of this calculation is the assumption of accurate estimates of the length

of the gonotrophic cycle. We have assumed that two or more blood-meals are required for the

first oviposition and that the temporal feeding pattern is not regular, and therefore, we fol-

lowed the method of calculations proposed by Garret-Jones and Grab [28]. Various studies

have estimated the gonotrophic cycle of An. darlingi to be 2–3 days [29, 30, respectively].

Recently, it was calculated to be 2.19 days in the rainy season and 2.43 in the dry season [31].

Calculations in our study were performed using the 2.19 day estimate based on the timing of

our An. darlingi collections (the rainy season).

Laboratory procedures

Individual An. darlingi were bisected between the head/thorax and abdomen and DNA was

extracted manually using the DNeasy Blood & Tissue kit (Qiagen). A PCR-RFLP protocol was

performed to detect the most common host in the area [32] for all mosquito abdomens in

Fig 2. Proportion of the domestic and wild animals in the study localities based on host censuses in

2013–2015. Additional animals seen frequently by the inhabitants were rats, toads, snakes and wild rodents.




2013–2015, except for a subsample (60%) of mosquitoes collected in LUP 2014 (due to a

extended sample size). In addition, samples were tested for Galliformes (Gallus gallus and tur-

keys; see census and proportion of chickens; Fig 2, S1 Table) following [33], rat and didelphis

[34], and monkey [35]. A subsample of the unidentified blood samples was sequenced for the

mitochondrial COI gene [36] and then compared with sequences in GenBank using BLASTn

(http://www.ncbi.nmln.nih.gov) or BOLD SYSTEMS v2.5 (http://www.barcodinglife.org). The

best match with identity of 95% or above was recorded.

Detection of Plasmodium infection was conducted using real-time PCR of the small subunit

of the 18S rRNA, with a triplex TaqMan assay (Life Technologies), as described in [37]. First,

DNA was extracted from each specimen of An. darlingi, then the RT-PCR was conducted on

pools of DNA of head/thoraces of five mosquitoes, and finally the pools were analyzed for

detection of P. vivax and P. falciparum. Specimens from positive pools were tested individually

to calculate infection rate (IR).

Data analysis

HBI was calculated as the proportion of mosquitoes fed on a specific host divided by the num-

ber of mosquitoes analyzed (mixed blood-meals were added to totals of each host). To adjust

the HBI, mosquitoes with unidentified blood-meals were excluded. This index was calculated

monthly in each locality and Chi-square (χ2) analyses were performed to compare statistical

differences temporally and among sites. Host data recorded in the census was used for the cal-

culation of the forage ratio (wi) [38, 39] and selection index (Bi) [40], to quantify the preference

of mosquitoes for available blood resources. The forage ratio for species i was calculated as

wi ¼ oipi

, where oi is the proportion of host species i in the blood-meals, and pi is the proportion

of available host in the environment. Forage ratios >1.0 indicate preference and< 1.0 avoid-

ance and selection of another host; ~1.0 means neither preference nor avoidance. The selection

index Bi was calculated with the formula Bi ¼wiPn

i¼1wi

, where wi is the forage ratio for species i

and n is the number of blood sources available.

Wind speed was measured at 6:00pm, 12:00am, and 6:00am each collection night in LUP,

CAH, and SEM in 2015. For each collection night, mosquito density was aggregated into four

3-hour collection periods (6-9pm, 9pm-12am, 12-3am, and 3-6am). The wind speed at 6:00pm

was assigned to the 6-9pm collection time, the wind speed at 12:00am was assigned to the

9pm-12am and 12-3am collection times, and the wind speed at 6:00am was assigned to the 3-

6am collection time. The mosquito density was plotted against wind speed for each collection

period at each location (n = 48 collection periods each for LUP and CAH, and 40 collection

periods for SEM) using the ggplot2 package in RStudio v0.98.1091 [41].

A null-model analysis was used to test whether An. darlingi feeding habits were random or

structured among the three villages, as in [36] and [42]. All specimens with identified blood-

meals from 2013–2015 for LUP, 2013–2015 for CAH, and 2014–2015 for SEM were included,

and specimens with mixed blood-meals were counted once for each host identified in the

blood-meal. We calculated a C-score comparing the blood- meal sources of mosquitoes from

the three villages using Ecosim 7.0 and we used the R bipartite package [43] to generate a host-

vector quantitative interaction network for the three localities, as in [36].

Results

Barrier screen mosquito collections

In 2013, all specimens caught on the screens were collected and identified to determine the

potential use of screens for collecting not only Anophelinae but also other Culicidae, potential



http://www.ncbi.nmln.nih.gov/

http://www.barcodinglife.org/

vectors of parasites and arboviruses. A total of 322 mosquitoes in LUP and 514 in CAH were

collected in 6 nights (72 h collection) (Table 1); 94.4% (304/18) of mosquitoes collected in LUP

and 89.7% (461/53) of all mosquito species in CAH were females. Anopheles darlingi com-

prised 78.9% and 61.5% of these collections in LUP and CAH, respectively, and Culex quinque-fasciatus was the second most common species identified in both localities (Table 1). Only one

additional species of anopheline, Anopheles forattini, was identified (in LUP).

With respect to screen position, in LUP 63.4% of the An. darlingi were collected on the side

facing the houses (In) and 36.6% on the side facing the creek (Out), although this difference

was not significant (Kolmogorov-Smirnov test; p = 0.4). On both sides of the screen, most of

the specimens were collected <1m from the ground (Below; Table 2) (range 76.5–90.2%). In

CAH, 61.8% of the mosquitoes were collected on the house side and 38.2% on the creek side,

and 93.1% and 84.5% (In and Out, respectively) were caught<1 m from the ground. No differ-

ences were found between LUP and CAH for side of the barrier screen. Only 1.62% in LUP

and 6.57% in CAH of the An. darlingi females were determined by visual inspection to be

blood-fed, with no differences between screen sides (Table 3).

In 2014, using multiple barrier screens per locality, a total of 4,593 An. darlingi females

were collected in LUP, 175 in CAH and 216 in SEM (Table 2). One specimen of Anophelesdunhami in LUP and eighteen Anopheles benarrochi B in SEM were also identified as in [14].

In LUP, no significant differences were detected between the sides of four screens tested inde-

pendently. However, when data were grouped over months there was a significant difference

between mosquitoes collected on the side of the houses (In) and creek/vegetation side (Out)

(Wilcoxon test; p = 0.0313). In CAH, the four barrier screens were not homogeneous, with sig-

nificant differences in number of mosquitoes collected from each side (K-S; In: p = 0.0082 and

Table 1. Number of each mosquito species collected in 2013 pilot survey in LUP and CAH (one barrier

screen/locality twice monthly from March to May).

Locality Species id N (females/males)

LUP

Anopheles darlingi 254 (246/8)

Culex quinquefasciatus 60 (51/9)

Mansonia indubitans/titillans 5 (4/1)

Psorophora cingulata 2 (2/0)

Anopheles forattinii 1 (1/0)

CAH

Anopheles darlingi 316 (304/12)

Culex quinquefasciatus 101 (63/38)

Mansonia indubitans/titillans 72 (72/0)

Mansonia humeralis 15 (15/0)

Culex coronator 6 (3/3)

Culex declarator 1 (1/0)

Culex theobaldi 3 (3/0)

doi:10.1371/journal.pntd.0005337.t001

Table 2. Percentage (N) of Anopheles darlingi collected above or below 1m on barrier screens in 3 localities by year.

LUP CAH SEM

Position 2013 2014 2015 2013 2014 2015 2014 2015

Above (>1m) 21.2 (40) 23.5 (1,095) 13.2 (135) 9.8 (32) 17.7 (31) 12.7 (30) 16.2 (35) 18.8 (44)

Below (<1m) 78.8 (148) 76.5 (3,576) 86.8 (885) 90.2 (295) 82.3 (144) 87.8 (205) 83.8 (181) 81.2 (233)




Out: p = 0.0356), and when In/Out were compared by month (K-S; p = 0.0022). There were

also significant differences between collections in LUP and CAH (K-S, p = 0.0336). In SEM,

captures in May (two screens) and in June (four screens), were not significantly different

between screens.

In 2015, in LUP, 1,019 female mosquitoes were collected, 233 in CAH and 277 in SEM.

Most specimens were captured resting < 1m from the ground with little variation among

years and sites (Table 2).

Differences in mosquito density by time of collection and side of barrier screen were tested

(Fig 3) with time of collection split into four three-hour periods (6-9pm, 9pm-12am, 12-3am,

and 3-6am). In both LUP and CAH in 2015, there was a significant difference in the distribu-

tion of mosquito collection location (side of screen) by time period (Kruskal-Wallis p< 0.0001

for both sites), with higher proportions of mosquitoes found on the In (facing house) side of

the screen from 9pm-12am and 12-3am than from 6-9pm and 3-6am. In LUP and CAH in

2013 and 2014, and in SEM in 2015, there was no significant difference in mosquito density by

time of collection (Kruskal-Wallis p>0.05).

Plots of mosquito density against wind speed for each locality in 2015 are shown in Fig 4.

Overall, there was a negative but non-significant correlation between mosquito density and

wind speed (Pearson’s r = -0.09, p = 0.3). The correlation between mosquito density and wind

speed was also negative in LUP (Pearson’s r = -0.25, p = 0.1) and SEM (Pearson’s r = -0.27,

p = 0.09), but was positive in CAH (Pearson’s r = 0.14, p = 0.34) (Fig 4).

To investigate the diurnal behavior of An. darlingi, barrier screen collections were per-

formed in LUP and CAH from January to June, and in SEM from May to June from 6AM to

6PM twice January-June 2015. In LUP a total of 59 An. darlingi were collected during this

period and female activity was reported from 6AM to 9AM and from 2PM to 5PM. In CAH,

the number of collected specimens was 23, with an activity similar to LUP. In SEM, 33 mosqui-

toes were collected, with an extension of the flying activity until 8AM, and beginning again in

the evening at 4PM. In LUP, 20.3%, in CAH, 34.8% and in SEM 54.5% of diurnal An. darlingispecimens were collected on the house side (In).

Table 3. Summary of proportion of An. darlingi visually blood-fed vs. blood-fed determined by molecular analysis, collected using barrier screens

in 3 localities from 2013–2015.

Visually blood-fed mosquitoes Identified blood-meal

In Out Total In Out Not id Total id

Site Year N % (N) % (N) % (N) % (N) % (N) % (N) % (N)

LUP

2013 246 0.81 (2) 0.81 (2) 1.62 (4) 56.8 (138) 35.4 (86) 7.8 (19) 92.2 (243)

2014 4,593 0.74 (34) 0.39 (18) 1.13 (52) 55.5 (1,159) 43.8 (914) 0.7 (15) 99.3 (2,084)

2015 1,019 6.96 (71) 1.47 (15) 8.43 (86) 50.5 (448) 47 (417) 2.5 (22) 97.5 (887)

CAH

2013 330 3.28 (10) 3.28 (10) 6.57 (20) 61.2 (202) 32.7 (108) 6.1 (20) 94 (330)

2014 175 6.85 (12) 1.15 (2) 8 (14) 70.8 (119) 17.2 (29) 12 (20) 94 (168)

2015 233 9.87 (23) 0.42 (1) 10.3 (24) 57.5 (133) 39.5 (91) 3 (7) 96.5 (231)

SEM

2014 216 0.92 (2) 0 (0) 0.92 (2) 70.9 (144) 25.1 (51) 4 (8) 96 (203)

2015 277 9.02 (25) 5.41 (15) 14.44 (40) 50.6(137) 47.6 (129) 5 (1.8) 98.1 (271)

Side of screen facing house = In; side of screen facing forest/water = Out.




Variation in parity and daily survival rate

A total of 583 An. darlingi females from LUP were dissected in 2014 (12% of the total) and 19

in CAH (11%); in 2015, n = 633 in LUP (62%) and n = 153 (65%) in CAH were dissected. The

monthly mean parity rate in LUP in 2015 was ~ 55% (range 45.6–66.7) and in CAH it was ~

51% (range 27.8–64.5) (Table 4). No significant differences were found between months or

between localities, although in February, the rate was slightly higher compared to June. Mos-

quito age in LUP in March—April 2014 was 7.47 and 14.21 days, respectively, whereas in 2015

it ranged from 14.21–23.90 days. In CAH, mosquitoes collected in March 2014 were estimated

to survive 14.98 days, and between 3.73–20.24 days in 2015 (Table 4).

Blood-meal source identification

Blood-meal source was determined for 4,417 An. darlingi females (S2 Table). A total of 3,214

mosquitoes from LUP, 729 from CAH and 474 from SEM were analyzed. Single-host blood-

meals were the highest percentage among the blood-meals detected (69.98%) and human was

the most common blood source (42.5%), followed by Galliformes (25.1%) and dog (1.42%; Fig

5). Only 4% of the samples could not be identified to blood-meal source. Multiple blood-meals

were found in 1,272 mosquitoes and accounted for 30% of the blood- meals, with 1,262 double

feeds in the three localities, and triple feeds (n = 10) only identified in LUP.

In total, seventy-three samples with non-identified blood-meal source by PCR-RFLP, were

sequenced for 16S ribosomal DNA [36] and mammalian cytochrome-b [32]. Only ten were

Fig 3. Proportion of An. darlingi collected on the in (facing house, blue) vs. out (facing forest/water, orange) side of

barrier screen by time of collection in LUP, CAH, and SEM, 2013–2015. *Significant difference in the distribution of mosquito

collection location by time period (Kruskal-Wallis p < 0.0001).




Fig 4. Correlation between density of An. darlingi on barrier screens and wind speed. Mosquitoes were

collected from 6pm-6am from January-June 2015 in CAH and LUP and May-September 2015 in SEM. Linear

regression of mosquito density on wind speed shown for each location (CAH: Pearson’s r = 0.14, p = 0.34;

LUP: Pearson’s r = -0.25, p = 0.1; SEM: Pearson’s r = -0.27, p = 0.09).


Table 4. Parity rate, daily survival and age of An. darlingi collected by barrier screens from LUP and

CAH, 2014–2015.

% Nulliparous

(N)

% Parous (N) % Gravid (N) Daily

survival

rate (P)

Age (days)

Site/

Year

2014 2015 2014 2015 2014 2015 2014 2015 2014 2015

LUP Feb - 10.7 (8) - 60 (45) - 29.3

(22)

0.95 - 19.42

March 25.4

(64)

14.3

(12)

61.5

(155)

66.7

(56)

13.1

(33)

19 (16) 0.94 0.93 7.47 14.21

April 8.5 (30) 11.9

(13)

37.5

(132)

52.3

(57)

54

(190)

35.8

(39)

0.96 0.94 24.65 17.24

May - 12.2

(18)

- 52 (77) - 35.8

(53)

0.94 - 16.89

June - 8.8 (19) - 45.6

(99)

- 45.6

(99)

0.96 - 23.90

CAH Feb - 12.9 (4) - 64.5

(20)

- 22.6 (7) 0.94 - 15.85

March 13.6 (3) 10.2 (4) 50 (11) 64.1

(25)

36.4 (8) 25.7

(10)

0.94 0.95 14.98 20.24

April - 17.6 (9) - 45.1

(23)

- 37.3

(19)

0.92 - 11.28

May - 44.4 (8) - 27.8 (5) - 27.8 (5) 0.76 - 3.73

June - 28.6 (4) - 57.1 (8) - 14.3 (2) 0.86 - 6.51




identified as of human origin with the 16S protocol, whereas 23 were consistent with human

for cytochrome-b.

The distribution of blood-meal source in An. darlingi presented little temporal or spatial

variation. Evaluation of the proportion of feeds on single different hosts showed that in LUP,

no significant differences between years were detected by one-way ANOVA analysis; paired

Wilcoxon-tests were not significant when comparing years 2013–2014 with 2015 or 2013 and

2014. In CAH, no significant differences between the years 2013–2014, 2014–2015 or among

the 3 years were found. In SEM, a non-parametric Mann-Whitney test was not significant

comparing 2014 and 2015. For locality comparison, data from the same years and different

localities were compared. In 2013, there were no significant differences between LUP and

CAH, and in 2014 and 2015 a one-way ANOVA test did not show differences between sites.

HBI was calculated monthly (S3 Table) and annually (Table 5) per locality. In 2013, no sig-

nificant differences were detected in LUP or CAH. Mean HBI per year was non-significant

among localities (LUP, CAH, SEM) and years 2014–2015.

The Forage Ratio and Host Selection Index were calculated, accounting for single and mul-

tiple blood-meals (Table 6). Humans were the preferred source, closely followed by Galli-

formes, in all three settings for both years. When the Forage Ratio was analyzed, the weight per

host was used instead of the numerical presence at the site [36] (S4 Table), Galliformes were by

far the preferred host, with humans as the second most favoured. For example in LUP, the

Fig 5. Proportion of blood-meal source, Anopheles darlingi collected by barrier screens in LUP, CAH and SEM in 2013–2015.




Galliforme forage ratio ranged from 10.35 to 17.96 and the human forage ratio from 0.58–

0.72. The null model test indicated that the mosquito feeding patterns were aggregated among

the localities, indicating that diet overlapped more than expected between the localities,

although this finding was only marginally significant (C-score: 0.33, p = 0.08). The quantitative

interaction network of blood-meal source by locality (Fig 6) supported patterns of organiza-

tion based on the above-mentioned trophic preferences (humans and Galliformes) from the

three mosquito populations (LUP, CAH, SEM).

Plasmodium mosquito infection

A total of 5,387, 362 and 455 mosquitoes in LUP, CAH and SEM, respectively, collected on

barrier screens, were tested for Plasmodium. The Infection rate (IR) of mosquitoes varied

among sites and seasons, ranging from 0.20–3.85 in LUP, 0.51–14.3 in CAH and 0–2.04 in

SEM (Table 7). A logistic regression model analysis determined that IR was significantly

higher in CAH (p = 0.02) and SEM (p = 0.003) vs. LUP. No specimens from the diurnal collec-

tions in the three localities (n = 116) were positive for P. vivax, independent of the collection

season.

Table 5. Summary of variation of An. darlingi Human Blood Index (HBI) per year and locality.

HBI

Year/Locality LUP (range) 95% CI of mean CAH (range) 95% CI of mean SEM (range) 95% CI of mean

2013 0.74 (0.71–0.76) (0.67–0.80) 0.57 (0.46–0.63) (0.33–0.80) - -

2014 0.72 (0.66–0.87) (0.67–0.78) 0.69 (0.63–0.77) (0.65–0.73) 0.67 (0.66–0.69) (0.48–0.86)

2015 0.65 (0.58–0.7) (0.61–0.69) 0.67 (0.6–0.77) (0.6–0.73) 0.79 (0.75–0.82) (0.69–0.88)


Table 6. Forage ratio (wi) and host selection index (Bi) of Anopheles darlingi in LUP, CAH and SEM from 2013–2015. Values of 1/n of the standard-

ized wi or Bi indicate no preference, below relative avoidance and >1/n relative preference.

Collection year Host abundance Forage ratio Selection index

(wi) (Bi)

Site/ Host blood meal 2013 2014 2015 2013 2014 2013 2014 2015 2013 2014 2015

(N) (N) (N)

LUP

Human 180 1412 602 432 432 1.61 1.24 1.25 0.53 0.13 0.09

Dog 12 61 20 52 77 0.89 0.30 0.23 0.29 0.03 0.01

Galliformes 77 1188 495 557 509 0.53 0.88 0.87 0.17 0.09 0.06

Pig - 15 10 0 1 - 5.70 9.02 - 0.60 0.69

Goat - 13 7 4 4 - 1.23 1.57 - 0.13 0.12

CAH

Human 224 116 157 910 910 1.09 0.89 0.79 0.08 0.06 0.31

Dog 7 10 2 35 33 0.89 2.12 0.28 0.06 0.15 0.11

Galliformes 113 73 148 596 478 0.84 1.07 1.43 0.06 0.07 0.57

Pig 2 4 - 4 3 10.58 9.35 0.78 0.69

SEM

Human - 137 212 - 212 - 1.36 1.35 - 0.55 0.36

Dog - 4 4 - 25 - 0.33 0.21 - 0.13 0.05

Galliformes - 79 125 - 227 - 0.73 0.74 - 0.30 0.20

Pig - - 1 - 1 - - 1.35 - - 0.36




Discussion

Ours is the first study to conclusively demonstrate that An. darlingi readily feeds on Galli-

formes. Overall, the feeding preference of An. darlingi in the Peruvian Amazon is more vari-

able than previous studies have assumed. In addition, a consistent pattern of blood-meal

Fig 6. Quantitative interaction network of An. darlingi blood-meal sources in SEM, CAH, and LUP.

Network is based on the analysis of blood-meal source for 4,417 An. darlingi females collected from 2013–

2015.


Table 7. Summary of Plasmodium detection in An. darlingi collected in all localities by barrier screen 2014 and 2015.

Year Locality Season1 # Collection Months Total Collected # inf. P. vivax IR

2014 CAH Rainy 6 157 2 1.27*

2014 CAH Dry 3 7 1 14.3*

2015 CAH Rainy 6 198 1 0.51*

2014 LUP Rainy 6 4356 132 0.30

2014 LUP Dry 3 26 1 3.85

2015 LUP Rainy 6 1005 2 0.20

2014 SEM Rainy 2 196 4 2.04*

2015 SEM Rainy 2 157 2 1.27*

2015 SEM Dry 3 102 0 0

1Rainy season: Jan-June, dry season: July-Dec2Two Plasmodium could not be identified to species.

* Logistic regression, CAH, P = 0.02; SEM, P = 0.003.




source was observed at each site every year of collection: mosquitoes feeding only on humans,

only on chickens, or on both hosts. This consistency could suggest the co-occurrence of differ-

ent subpopulations within a metapopulation, with local adaptation as the main driving force.

A single metapopulation was initially detected in An. darlingi in the Iquitos area with

AFLPs [44] and microsatellite markers [45]. However, using 2x the number of microsatellites,

a population replacement event was detected between 2006 and 2012 and two subpopulations

were detected, one significantly more prevalent in highway compared with riverine habitat

[20]. This recent genetic structure could explain some of the heterogeneity in feeding prefer-

ences of An. darlingi among localities [45, 46]. Additional studies, focused on intrinsic host

preference, vector density and social practices of the human population might elucidate

the basis for the described behavior and whether some An. darlingi populations are under

selective pressure for host preference or whether this pattern is strongly correlated with host

availability.

Similar HBI across the dry and rainy seasons and between populations infers that mosqui-

toes maintain their host preference behavior independent of local ecological conditions. In an

earlier investigation of HBI of An. darlingi in riverine villages in Amapa State, Brazil [18],

researchers reported high among-village variance (HBI 0.131–0.435) and ~10% of mixed

blood-meals overall, mainly from cattle and pigs. In contrast, in our study, there was virtually

no variance in HBI among localities, HBIs were higher (0.58–0.79) and ~30% of blood-meals

were mixed, with Galliformes as the primary alternate host. Because HBI is an integral parame-

ter of the vectorial capacity formula (the daily rate of malaria transmission from a single

infected human, assuming every bite from an infected mosquito leads to transmission) [2], our

data suggest that An. darlingi is a more effective vector in the peri-Iquitos area compared with

Amapa state, Brazil. Curiously, in Tanzania, An. arabiensis avoids, and may be repelled by, the

volatiles of chickens [47]. Subgenera Nyssorhynchus (An. darlingi) and Cellia (An. arabiensis)were estimated to have diverged ~94 million years ago [48]; therefore their olfactory responses

are expected to have evolved differentially.

The present study provides evidence of the successful use of barrier screens to collect

blood-fed An. darlingi mosquitoes in Amazonian Peru. Initially, in 2013, we conducted pre-

liminary barrier screen collections with Procopack aspirators in LUP and CAH from 5 to 8

AM for 6 days/collection in March-May in at least 10 houses each time, but only one An. dar-lingi specimen was caught. Interestingly, in Iquitos the Procopack effectively collected indoor

resting Culicidae including Aedes aegypti and Culex pipiens complex [49]. One explanation for

our failure to find An. darlingi using the Procopack despite extensive searching could be due to

its singular resting and biting behavior in this region.

Anopheles darlingi resting behavior varies across its range [50]: in Venezuela, Guyana [51]

and in Brazil, in Matto Grosso and in southern Amazonas [52, 53] it rests during the day inside

houses (endophily). In contrast, in Suriname, using exit traps, a peak departure from the dwell-

ing was observed at sunrise [54] and in Brazil An. darlingi was resting indoors only at night

[55]. In Amapa state, Brazil, resting mosquitoes were collected after sunrise (6AM-7AM)

under houses and in peridomestic vegetation [18]. In French Guiana, no resting An. darlingiwere collected indoors after pyrethroid spray, from pit-shelters or in the shade in the perido-

mestic area [56]. In our study, overall differences detected between screen sides may reflect the

relative nearness of screens to houses, resulting in the interception of a higher proportion of

blood fed An. darlingi leaving the peridomestic area, compared with questing females, entering

the village from numerous resting and/or breeding sites. In CAH, we hypothesize that addi-

tional differences among screens and between months could result from a much smaller popu-

lation of An. darlingi intercepted in this village. Our results constitute a major accomplishment:



the use of barrier screens in this setting to overcome the difficulty of performing host-indepen-

dent sampling for determining blood-meal sources.

The success of individual mosquito blood-meal identification in this study (range of 92.2–

99.3%), was remarkably high when compared to visually blood-fed mosquitoes (0.92%-

14.44%). When analysis is restricted only to the latter, information from partial blood-meals

or partially digested blood is missed, leading to underestimation of the proportion of host

sources (up to 18.7%); hence, a miscalculation of HBI [57]. One limitation of our study was the

lack of identification of potential wild animal hosts; use of novel targeted high-throughput

sequencing [58] would rectify this.

In LUP, the age of the mosquito population at each time point is enough to sustain the spo-

rogonic cycle of P. vivax (range 7.24–9.13 days; calculated by the Moshkovsky method in [31]),

whereas in CAH the population is, in general, younger, but with non-dangerously aged mos-

quitoes only in May and June. The proportion of young females might be explained by differ-

ential dispersal and aggregation of different age classes of An. darlingi populations, as

previously reported for An. farauti in Papua New Guinea [59]. Use of 2.19 days of the gono-

trophic cycle [31] could have produced a miscalculation in the age parameter. For instance,

gravid females may experience delays while searching for suitable oviposition sites or there

could be variation in extrinsic environmental conditions within this population of An. darlingi[60]. Because of the natural development of the parasite within the mosquito, a longer life-

span is related to a higher potential to transmit malaria [61]. Parity is also associated with sea-

sonality, i.e., mosquitoes generally survive longer during the rainy season [62,63], but see [64].

Overall, our study provides unreported information of the blood-meal preferences of An.

darlingi in the peri-Iquitos area, which will be the base-line to compare potential changes in

the behavior of these mosquito populations. HBI, together with other malaria metrics such as

HBR or EIR, should be taken into consideration for surveillance and epidemiological studies

of malaria transmission.

Supporting information

S1 Fig. Construction and set up of the barrier screens in Iquitos, Peru; (A, B, C, F): 2 m

high and 15m long. Screens were examined hourly by flashlight and resting mosquitoes cap-

tured by aspiration (D, E).

(TIF)

S1 Table. Census of domestic and wild animals in the study localities 2013–2015. Rats,

toads, snakes and wild rodents were other animals frequently observed by the inhabitants.

(DOCX)

S2 Table. Summary of An. darlingi blood-meal sources per year per locality.

(DOCX)

S3 Table. Monthly variation of Human Blood Index (HBI) for An. darlingi in three sites.

(DOCX)

S4 Table. Forage ratio (FR) of An. darlingi using host biomass. Mean weight of hosts was:

human (65kg), dog (25 kg), chicken (1.5kg), turkey (13.1 kg), pig (90kg), goat (45 kg).

(DOCX)

S1 Dataset. Summary of mosquito collections by barrier screens methodology. Collection

site and dates of collection, mosquito species identification and side and height of the barrier

screens are designated for each mosquito used in the analysis.

(XLSX)



http://journals.plos.org/plosntds/article/asset?unique&id=info:doi/10.1371/journal.pntd.0005337.s001






S2 Dataset. Mosquito blood-meals. The file shows, for each mosquito analyzed, the source of

blood-meal identified.

(XLSX)

Acknowledgments

We would like to thank Eliseo Ramirez, Jose Manuel Reyna, Victor Pacaya, David Arimuya,

and Hercules Maytahuari for their assistance in the field. We appreciate the enthusiastic sup-

port of the communities of Cahuide, Lupuna and Santa Emilia (Loreto Department). We are

grateful to Direccion Regional de Salud (DIRESA, Iquitos, Loreto) for collaboration and facili-

tating logistics in Loreto. We are grateful to the Applied Genomic Technologies Core at the

Wadsworth Center, New York State Department of Health for the sequencing of the samples.

Author contributions

Conceptualization: MM JEC.

Formal analysis: MM SAB CP.

Funding acquisition: JEC JMV.

Investigation: MM MPS SAB CP AM CTR JEC.

Methodology: MM JEC SAB MPS CP.

Resources: JEC JMV.

Visualization: MM SAB CP JEC.

Writing – original draft: MM JEC.

Writing – review & editing: MM JEC MPS SAB CP AM CTR JMV.

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